PROJECT SUMMARY Pulmonary vascular disease (PVD) is an important source of morbidity and mortality in patients with congenital heart disease (CHD). The natural history of PVD in these patients reveals the important pathophysiologic differences associated with abnormal pulmonary blood flow (PBF) and pressure. Patients with cardiac defects that expose the pulmonary vasculature to increased flow with a direct pressure stimulus from the systemic ventricle develop PVD with greater incidence and severity than patients with defects resulting in increased PBF alone. Pulmonary endothelial cells (EC) are integral mediators of disease, due to their exposure to these normal and abnormal hemodynamic (mechanical) forces including shear stress, hydrostatic pressure, and cyclic strain. Our laboratory has developed two distinct, clinically relevant models of CHD in fetal lambs: (1) left pulmonary artery (LPA) ligation that primarily results in increased PBF to the right lung; and (2) aortopulmonary shunt placement that results in increased PBF and pressure. Our preliminary data demonstrate that at 4-6 weeks of age, model lambs manifest distinct aberrations in endothelial cell signaling and vascular function. For example, RNAseq analysis performed on primary pulmonary artery endothelial cells (PAEC) from each lamb model demonstrates markedly distinct gene expression patterns, and studies in isolated vessels demonstrate disparate alterations in vascular reactivity. Moreover, we have generated novel in vivo and in vitro data demonstrating that the additive effects of the biomechanical forces?fluid shear stress and pressure induced cyclic stretch?cause perturbations in cellular signaling pathways that result in endothelial dysfunction (eNOS uncoupling), metabolic reprogramming (ROS driven HIF-1a, and c-MYC activation), and a hyper-proliferative, anti-apoptotic, endothelial cell phenotype. Based on these data, the overall hypothesis we will test in Project #1, is that the distinct mechanical forces associated with increased PBF compared to increased PBF and pressure, induce patterned alterations in gene expression and vascular function that underlie the incidence and progression of PVD associated with CHD. Specifically, we hypothesize that flow-alone maintains NO signaling through ATP- dependent hsp90 activity and c-MYC-mediated glutamine anaplerosis. The addition of pressure induced cyclic stretch, however, leads to HIF-1? driven Warburg metabolism and EC hyper-proliferation via increases in mitochondrial (mt)-ROS production, but at the expense of ATP-dependent hsp90 activity and NO signaling. This overall hypothesis will be tested in three inter-related, but independent, Specific Aims. As current PVD treatment approaches are based on disease severity as opposed to underlying pathobiology, the successful completion of the proposed studies may lead to targeted therapeutic approaches for PVD 2 to CHD, as well as inform other types of PVD, in which abnormal mechanical forces participate in disease progression.